Supplementary Information
Effects of Bushen Tianjing Recipein a rat model of tripterygium glycoside-induced premature ovarian failure
Xiaofeng Xu1, Yong Tan2, Guorong Jiang1, Xuanyi Chen1,*, Rensheng Lai3, Lurong Zhang1, Guoqiang Liang1
1Department of Obstetrics and Gynecology, Suzhou Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu Province 215009, China
2 Department of Gynecology, the No.1 Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu Province 210029, China
3 Department of Pathology, Suzhou Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu Province 215009, China
*Corresponding Author: Xuanyi Chen, Professor of Medicine
Affiliation: Department of Obstetrics and Gynecology, Suzhou Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu Province 215009, China
Address: No. 18 Yangsu Road, Gusu District, Suzhou, Jiangsu Province 215009, China
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Materials and Methods
Chemicals and reagents
The reference standards of catalpol, paeoniflorin, morroniside, and argininewere purchased from the National Institutes for Food and Drug Control (Beijing, China). The high performance liquid chromatogram (HPLC) column was from Thermo Fisher Scientific (HypersilTM C18 Column, Waltham, MA, USA). The ultraviolet detector was from Hitachi (U-3300, Tokyo, Japan). All other solvents (HPLC grade) were purchased fromSigma-Aldrich, China (Shanghai, China).
Procedures of high performance liquid chromatogram
The procedures of high performance liquid chromatogram (HPLC) were as follows. First, 1 g BTR was dissolved in 80 % methanol (v/v) with the aid of ultrasonication for 30 min. Then, the resulting solution was filtered using a membrane filer (pore size = 0.25 μm, Merck Millipore, Billerica, MA, USA), and 10 μl filtered solution was injected into the HPLC system (Agilent1100 HPLC System, Santa Clara, CA, USA) for analysis. The reference standards of catalpol, paeoniflorin, and morronisidewere also dissolved in 80% methanol at concentration of 50 μg/ml. The HPLC conditions for catalpol were: the mobile phase,0.1% phosphate buffer; the flow rate,1.0 ml/min; the wavelength of detection,210 nm; and the column temperature, 25 ºC. The HPLC conditions for paeoniflorin were: the mobile phase, methanol-water-phosphate buffer (28: 72: 0.1, v/v); the flow rate, 1.0 ml/min; the wavelength of detection, 230 nm; and the column temperature, 25 ºC.The HPLC conditions for morroniside were: the mobile phase, methanol-water-phosphate buffer (28: 72: 0.1, v/v); the flow rate, 1.0 ml/min; the wavelength of detection, 250 nm; and the column temperature, 25 ºC.The contents of catalpol, paeoniflorin, and morroniside were calculatedfrom the area under the peak, at the base of their individual external standards.
Procedures of thin layer chromatography
Thin layer chromatography (TLC) was used to detect the presence of arginine, an active component of Testudinis Carapax et Plastrum, in BTR according to the method described in previous literature [1]. Briefly, standard arginine solution (1 mg/ml) was preparedin 0.01 M phosphate buffer (pH 8.0). Then, 1 µlBTR and 1 µl standard arginine solution were spotted on the TLC plates (Silica gel 60G F₂₅₄, Merck Millipore, Catalog No. 1003900001).Plates were air-dried and then developed using n-propanol-water (70: 30, v/v) as the mobile phase. After development, plates were dried and visualized with0.25% ninhydrin in acetone.
References
1.Sen S, Sarkar S, Kundu P, Laskar S. Separation of amino acids based on thin-layer chromatography by a novel quinazoline based anti-microbial agent. Am J Analyt Chem. 2012; 3: 669-74.
Figure Legends
Supplementary Figure 1. The representative HPLC chromatogram ofcatalpolin BTR. A: The reference standard of catalpol; B: BTR sample (batch No.:BSTJ001).
Supplementary Figure 2. The representative HPLC chromatogram ofpaeoniflorinin BTR. A: The reference standard of paeoniflorin; B: BTR sample (batch No.: BSTJ001).
Supplementary Figure 3. The representative HPLC chromatogram ofmorronisidein BTR. A: The reference standard of morroniside; B: BTR sample (batch No.: BSTJ001).
Supplementary Figure 4.The representative TLC chromatogram ofargininein BTR. Lane A: the reference standard of arginine; lane B: 0.01 M phosphate buffer (pH 8.0) as negative control; lane C:BTR sample (batch No.: BSTJ001).
Supplementary Figure 5. Quantitative analysis of numbers of follicles in BTR treated animals.For each animal, 10 high power fields (HPFs) were randomly chosen from five sections for counting the number of follicles (both primordial and primary). Each dot represents the average value across these HPFs. Bars and error bars are means and SD, respectively. All statistical analyses were performed using ANOVA followed by Tukey’s post-hoc test. NS indicates not significant.
Supplementary Table 1. HPLC and TLC analyses of three batches of BTR
Batch No. / BSTJ001 / BSTJ001 / BSTJ001 / Average / RSD (%)Content of catalpol
(mg/10 g BTR) / 55 / 59 / 56 / 57 / 3.7
Content of paeoniflorin
(mg/10 g BTR) / 220 / 237 / 241 / 233 / 4.8
Content of morroniside
(mg/10 g BTR) / 62 / 61 / 65 / 63 / 3.3
TLC analysis of arginine / Positive / Positive / Positive / N/A / N/A
BTR, Bushen Tianjing Recipe; RSD, relative standard deviation
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